Tumor‐expressed microRNAs associated with venous thromboembolism in colorectal cancer

Abstract Background Colorectal cancer patients have an increased risk of developing venous thromboembolism (VTE), resulting in increased morbidity and mortality. Because the exact mechanism is yet unknown, risk prediction is still challenging; therefore, new biomarkers are needed. MicroRNAs (miRNAs) are small, relatively stable RNAs, that regulate a variety of cellular processes, and are easily measured in body fluids. Objective The aim of this study was to identify novel tumor‐expressed miRNAs associated with VTE. Methods In a cohort of 418 colorectal cancer patients diagnosed between 2001 and 2015 at the Leiden University Medical Center, 23 patients (5.5%) developed VTE 1 year before or after cancer diagnosis. Based on availability of frozen tumor material, tumor cells of 17 patients with VTE and 18 patients without VTE were isolated using laser capture microdissection and subsequently analyzed on the Illumina sequencing platform NovaSeq600 using 150‐bp paired‐end sequencing. Cases and controls were matched on age, sex, tumor stage, and grade. Differential miRNA expression was analyzed using edgeR. Results A total of 547 miRNAs were detected. Applying a 1.5‐fold difference and false discovery rate of <0.1, 19 tumor‐miRNAs were differentially regulated in VTE cases versus controls, with hsa‐miR‐3652, hsa‐miR‐92b‐5p, and hsa‐miR‐10,394‐5p as most significantly downregulated. Seven of the 19 identified miRNAs were predicted to regulate the gonadotropin‐releasing hormone receptor pathway. Conclusion We identified 19 differentially regulated tumor‐expressed miRNAs in colorectal cancer‐associated VTE, which may provide insights into the biological mechanism and in the future might have potential to serve as novel, predictive biomarkers.


Essentials
• microRNAs are promising biomarkers for venous thromboembolism (VTE) in patients with cancer.
• microRNA profiling was performed on colorectal cancer samples from patients with and without VTE.
• 19 tumor-expressed microRNAs were differentially regulated in patients with VTE.
• 7 of 19 identified microRNAs predicted to regulate Gonadotropin-releasing hormone receptor pathway.

| INTRODUC TI ON
Cancer patients have a 9-fold increased risk of developing venous thromboembolism (VTE) compared with the general population in the first year after diagnosis. 1,2 The risk is determined by several cancer-related factors, including chemotherapy, surgery, tumor type, and stage. 2 Colorectal cancer patients have an intermediate risk of VTE with a 6-month incidence rate of 5 per 100 person-years after diagnosis. 3 Because it is the third most diagnosed cancer type worldwide, with 1.8 million new cases annually, the absolute number of VTE occurrences in these patients is high. 3 Cancer patients at high risk of VTE may benefit from thromboprophylaxis. 4,5 Currently, the Khorana risk score, including clinical and laboratory variables, is the only model endorsed for clinical use. 6,7 However, its performance is suboptimal; therefore, an improved risk stratification is urgently needed. 8  level. 9 miRNAs are expressed intracellularly but are also secreted and detectable in blood and other body fluids. Importantly, miRNAs are relatively stable because of their resistance to nuclease digestion, leading to high reproducibility when measured. 9,10 Therefore, miRNAs appear to be suitable biomarkers, particularly when assayed in relatively accessible body fluids. 9 In contrast, obtaining tumor material from miRNAs is more complex, but the tumor-expressed miRNA profile may better elucidate which tumor-intrinsic pathways contribute to CAT. Although it was recently shown that certain miRNA plasma levels are associate with CAT development, no studies to date have explored associations between tumor-expressed miRNAs and CAT. [11][12][13][14] In this nested case control study, we have compared tumor expressed miRNA profiles from colorectal cancer patients with VTE to those without VTE, which may lead to new biological insights and potentially novel biomarkers for CAT.

| ME THODS
Between January 2001 and December 2015, 418 colorectal cancer patients were identified who underwent curative or palliative surgery at the Leiden University Medical Center (LUMC; Figure 1). VTE events were retrospectively assessed from hospital records and occurred in 23 patients (5.5%) 1 year before or after cancer diagnosis. 15   To exclude any effect of cancer-related treatment on VTE, a subgroup analysis was performed on VTE patients in the year before cancer diagnosis (four pairs).
Data were analyzed using R-studio version 4.0.2. The Benjamini-Hochberg method was used to adjust p values for a false discovery rate (FDR). As the aim here was an exploratory screening, a predetermined statistical significance cutoff of <0.1 was used and a fold change (FC) of 1.5 was considered to represent a meaningful biological effect.  Table S1 for more detailed information on the individually matched patient couples).

TA B L E 2 (Continued)
One miRNA in our panel, miR-483-5p, was previously identified in a noncancer thrombosis setting. miR-483-3p, its minor miRNA arm, was upregulated in endothelial cells from deep vein thrombosis patients compared with control cells and its overexpression diminished thrombus resolution in vivo. 18 However, in the current study, To date, two studies addressed a role for miRNAs in CAT. In pancreatic ductal adenocarcinoma and distal extrahepatic cholangiocarcinoma patients, 11 plasma miRNAs associated with VTE, but none of these were identified in our study. 11 In another study, six upregulated plasma miRNAs from glioma patients predicted the risk of early postsurgical pulmonary embolism. 12 Interestingly, miR-363-3p, also identified in the current study, was one of the six. 12 Furthermore, in a conference abstract presented by the same research group, miR-363-3p was also increased in plasma from lung CAT patients. 13 It is tempting to speculate that the increased levels of miR-363-3p observed in plasma from CAT patients are a result of increased expression and secretion of miR-363-3p by tumor cells. However, it remains to be investigated whether miR-363-3p is also upregulated in plasma from colorectal cancer patients with VTE, and whether miR-363-3p is also differentially regulated in pancreatic, cholangiocarcinoma, glioma, and lung cancer cells of VTE patients. In addition, increased miR-363-3p plasma levels might be caused in part by higher expression and secretion by noncancerous cells as well, reflecting a more general hypercoagulable state. In cancer, miR-363-3p was described to regulate integrin and ERK1/2-signaling, but whether these pathways are used to exert thrombogenic effects is unknown. 19,20 Besides miR-363-3p, there was no overlap of identified miRNAs between the current study and those described previously. This could be explained by two major differences between our study and the two published reports, which were tumor type (colorectal cancer vs. glioma, pancreatic, and cholangiocarcinoma) and type of sample (tumor tissue vs. plasma). Regardless of differences in study design, the finding that miR-363-3p was upregulated in plasma from glioma and lung cancer patients and in colorectal tumor tissue in patients with VTE, is of interest and suggestive of a causal role for miR-363-3p in CAT.
To explore pathways through which the identified miRNAs may exert prothrombotic effects, the top three predicted pathways of all miRNAs were identified using miRPathDB v2.0. and PANTHER pathways algorithm ( (~90% of the 19 identified miRNAs) true-positive. If the more stringent FDR cutoff level of 0.05 was applied, hsa-miR-3652, hsa-miR-92b-5p, and hsa-miR-10,394-5p would still be considered significant.
Nonetheless, functional validation of all of these miRNAs is warranted in in vitro, ex vivo, and in vivo CAT models.
Last, data on race or ethnicity was not available in our cohort; the results from this cohort need to be cautiously interpreted when translated to a cohort in a country with a different distribution of race/ethnicity.
In conclusion, a tumor-expressed miRNA profile associated with CAT was established, which may contribute to hypercoagulability in colorectal cancer. As follow-up, external validation of this profile is first needed in a larger colorectal cancer cohort and subsequently experimental validation. Moreover, it remains of utmost interest to investigate whether these tumor-expressed miRNAs are also differentially regulated in plasma of colorectal cancer patients so that they could be used as potential CAT plasma biomarkers.

ACK N OWLED G M ENTS
This work was supported by the Dutch Cancer Society (#13189) and Dutch Digestive Foundation (#SK 18-17). We acknowledge Dr.
Andrew Stone, Drs. Yvonne Jongejan, and Drs. Mark Smeets for commenting on the manuscript.

R EL ATI O N S H I P D I SCLOS U R E
The authors have no conflicts of interest.